The ability to directly visualize the device tissue interface during cardiac ablation procedures is invaluable. The utilization of Visible Heart® methodologies has allowed for investigations of cardiac ablative procedures, specifically this procedural application and associated complications that may arise within the various cardiac chambers. For instance, the explosive steam pop phenomenon was visualized inside the atrium to demonstrate the intense release of energy and potentially damaging particulates. Furthermore, additional infrared imaging investigations on the epicardial tissue temperatures were conducted to further improve our understanding of the temperatures, which the nearby phrenic nerve may experience during a performed clinical procedure. Additionally, a typical cryoballoon ablation procedure was visualized from the transseptal puncture to the difficulty clinicians face when positioning balloon. The information gained from these investigations may provide important insights to both clinicians performing such procedures as well to device designers.
In vitro Reanimation of Isolated Human and Large Mammalian Heart-
Lung Blocs
Submitted to BMC Physiology, in review.
Ryan P. Goff, PhD1,2, Brian T. Howard, MS1,2, Stephen G. Quallich, BS1,2, Julianne H. Spencer, PhD1,2, Tinen L. Iles, BS2, Paul A. Iaizzo, PhD2
1
Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN
2
Department of Surgery, University of Minnesota, Minneapolis, MN
Preface
Although this study does not directly examine cardiac ablation procedures, it provides the background required to generate a heart-lung bloc model that has numerous benefits for such studies. The addition of the lung(s) provides native pulmonary vein ostium that can then be invaluable for studying the device/tissue interface. This study describes the reanimation of both swine and human heart-lung blocs; the development of this methodology also allowed for the following two studies to be conducted. For example, epicardial infrared imaging to observe surface temperatures during pulmonary vein isolation would not be representative of the clinical scenario. The addition of the lungs to our reanimation procedure fabricated a more robust model that accurately represents the native anatomy and physiology, especially when performing cryoballoon ablation procedures.
I was responsible for data and statistical analysis of swine and human heart specimens. In addition, all of the contributors aided with the reanimation of these cardiac specimens. The lab is still using these techniques today, especially when representative pulmonary vein anatomy is required.
Summary
Background: In vitro isolated heart preparations are an invaluable tool for the study of
cardiac anatomy, physiology, and device testing. Such preparations afford investigators a high level of control, independent of host or systemic interactions, and high throughput if desired. Here we present that isolated human and swine preparations with the lung(s) attached are particularly valuable for the study of device-tissue interaction and anatomy. Additionally we detail our laboratory’s experience with developing these methodologies for heart/lung bloc studies
Methods and Results: Four human and 18 swine heart-lung preparations were procured
using techniques analogous to those of cardiac transplant. Specimens were then rewarmed and reperfused using modifications of a previously developed apparatus and methodologies by our laboratory. Positive pressure mechanical ventilation was also employed, and epicardial defibrillation was applied to elicit native sinus rhythm after rewarming. Videoscopy, fluoroscopy, ultrasound, and infrared imaging were performed for anatomical and experimental study. Systolic and diastolic pressures observed for human and swine specimens, respectively, were 68/2±11/7 and 74/3±17/5 mmHg, with heart rates of 80±7 and 96±16 bpm. High resolution imaging within functioning human pulmonary vasculature was obtained among other anatomies of interest. One specimen elicited poor cardiac performance post-defibillation.
Conclusion: We report the first dynamic images of the pulmonary vasculature during
cardiopulmonary function in isolated reanimated heart-lung blocs. This experimental approach provides unique in vitro opportunities for the study of medical therapeutics applied to both human and large mammalian heart-lung specimens.
Key Words: heart-lung bloc; device-tissue interaction; pulmonary vasculature; isolated
Introduction
In vitro isolated heart preparations have been a cornerstone of cardiac research since Langendorff’s original methodology was described in the 1890s [141]. The benefits of isolated heart research are numerous and can be remarkable depending on the investigator’s goal. Isolated hearts offer a high degree of control over the system including, but not limited to: perfusate selection, flow control, and pre- and after-load variability. For a thorough historic summary of these experimental models the reader is referred to a review by Hill et al [142]. Furthermore, numerous pharmacological studies using such approaches can help elucidate the direct action of agents on the isolated cardiac tissues, i.e., while avoiding systemic interactions of other agents or breakdown products (e.g., cardiac-nervous system, hepatic metabolism) [143].
Additionally, high-throughput cardiac perfusion systems can be designed, or now even purchased off the shelf, in which multiple small mammalian hearts can be experimented on simultaneously. Isolated heart preparations have garnered notable insights to mechanisms of arrhythmias[144] and have been reviewed elsewhere[145]. Depending upon the system configuration, a wide range of equipment and modalities are available to the investigator including: electrophysiologic monitoring and stimulus, ultrasonography, ultrasonic stimulation, fluoroscopy, infrared thermography, direct visualization via videoscopes, and anatomical mapping systems. Furthermore, the utilization of large mammalian isolated hearts allows for critical pre-clinical testing of device-tissue interactions in an environment highly similar to actual human anatomy and physiology, if the proper animal model is selected for investigation [146]. Comparative imaging of normal versus pathologic conditions, or interspecies comparisons, to determine optimal approaches, models, and designs are critical to development of novel therapeutics[147]. To the medical device designer, engineer, or clinician, these insights have proven to be of high educational value[148,149].
their native ostia are of interest in the context of pulmonary vein isolation ablation treatments for atrial fibrillation. Heart-lung preparations have been utilized previously to elucidate the release of atrial natriuretic peptide10 and expand the pool of lung transplants to non-beating donors [150]; they have also been used in numerous pharmacologic studies. Interestingly, the first heart-lung preparations are often attributed to Knowlton and Starling [151], however their work acknowledges the methods of Martin [152] which were presented in lecture at Johns Hopkins in 1883. The first publication by Martin of his heart-lung bloc preparation was released in 1881 [153], therefore predating Langendorff’s work by fourteen years. In short, this preparation cannulates in situ the superior vena cava and one of the branches coming off the aortic arch. A closed loop is created by which pressure can be monitored, a compliance chamber is incorporated, and pre- and afterloads are varied.
It is also possible for human hearts from non-viable organ donors to be successfully reanimated using an isolated experimental apparatus [142]. The Visible Heart® methodologies have been previously described by our laboratory [154], but more recently we have expanded these experimental approaches to incorporate whole large mammal heart-lung blocs, including both human and swine studies. To the authors’ knowledge, this is the first description of large mammalian heart-lung blocks being used to achieve dynamic imaging in the pulmonary vasculature. The goal of the current study is to determine feasibility and characterize viability of large mammalian heart-lung preparations.
Methods
The technique developed by our laboratory has been used successfully to reanimate human and swine hearts with right, left, or both lungs attached and functioning. Swine studies were approved by the Internal Animal Care and Use Committee at the University of Minnesota. Human hearts were approved for study by the Human Subject Committee Institutional Review Board. Consent for use of the hearts for research purposes was
received from the donors’ family members before explantation via LifeSource (St. Paul, MN, USA).
The detailed procurement procedure has been described previously [142,154]. Briefly, a median sternotomy was performed and an aortic root cannula implanted for delivery of cardioplegia. The inferior vena cava (IVC) was ligated and, just prior to cardioplegia delivery, the IVC for human preparations was removed with the liver if it was being recovered for transplant, and the superior vena cava (SVC) and aorta were cross-clamped. Cardioplegia was then delivered under pressure to rapidly cool and arrest the heart. The heart and lungs were then dissected and the heart-lung bloc removed by transection of the major vessels, trachea, and esophagus. The human specimens were then transported on ice to the laboratory within 6 hours of cross-clamp depending upon logistics of transportation of viable organs to recipients. The human heart-lung specimens were non- viable cardiac donors (e.g., unknown cardiac arrest period, cardiac disease). An analogous procedure was performed on swine hearts in our laboratory (mean animal weights of 84±14kg; n=18) using two liters of 4°C St. Thomas’s cardioplegia for induction of cardiac arrest. We have typically performed these studies with just one lung attached, but the method has been utilized to include both lungs. Preparations with only one lung allow cannulation of the non-utilized pulmonary vein, which may be used to access the left atrium for imaging or device introduction.
Upon arrival of human (or after explantation of swine) specimens, hearts were placed in an ice slurry of modified Krebs-Henseleit buffer while cannulation of the great vessels was performed (i.e., IVC, SVC, and aorta). If a one-lung preparation was desired, the left/right pulmonary veins and artery were dissected from the left/right lung, and the lung was removed. These vessels were cannulated as well, and a hemostasis valve was fitted for access. If both lungs were desired in the preparation, the pulmonary trunk was cannulated to allow control of the buffer flow, either directing all flow through the lungs or allowing some flow to the reservoir (i.e., a parallel path through the lungs and to the
control flow through the airway. Preparations were ventilated at a respiration rate of 11- 15 per minute and a volume of 150-250 milliliters per lung.
The heart-lung blocs were then connected to the apparatus described in detail previously [154] that was adapted for such use. A schematic of this system can be found in Figure 35. The system was altered to vary the aforementioned parameters of other isolated heart research systems and functioned in either partial or four-chamber working mode. Partial working mode is similar to a Langendorff apparatus function, but fluid flow continues through an isolated lung (i.e., the right heart continues to function). The system utilized a cardiovascular bypass oxygenator and heated water jacketed fluid reservoirs to maintain the proper physiologic environment. The preparations were cradled on custom sized soft foam cushions to support the tissue. Seven to eight liters of modified Krebs-Henseleit buffer were contained in the system and buffer changes of approximately four liters were performed regularly to wash out metabolites and maintain visualization as desired.
Figure 35. (Top right) External view of human heart 277 in systole and attached to the system. (Center) Flow diagram for a functional heart and lung reanimation consisting of: (1) a respirator connected to the cannulated trachea and thus attached to the lung(s), (2) a pre-load chamber for the right side of the heart, (3) an aortic after-load chamber which mimics the resistance that the left ventricle works against, (4) a left pre-load chamber employed when only one lung is present, (5) an oxygenator reservoir for pooling fluid expelled by any cannulated branch of the pulmonary artery, (6 & 7) fluid pumps to maintain the pre-load pressures, (8) hemostasis valves that allow access for delivery of cameras, instruments, and assorted devices, (9) valves that may also be used to redirect flow as physiologically appropriate, while (10) cannulation of the pulmonary vein(s) are shown here for a right lung preparation, but are absent or translated when either both lungs or the left alone respectively are used.
Once the specimen was re-warmed to 37°C, dobutamine was added to the system and the heart was defibrillated with 34 joules of energy supplied by a programmer-analyzer unit
(#88345 Medtronic, Inc., Minneapolis, MN, USA) via a pair of external patches (#6721, Medtronic, Inc.) placed epicardially above and below the ventricles. These hearts generally began beating in native sinus rhythm after a single defibrillation. It should be noted that one human heart developed heart block at two hours post-reanimation, and was then paced by a temporary pacing lead at 60 beats per minute; all specimens could be paced as desired. Hemodynamics of the left and right ventricle were recorded by Utah Medical pressure transducers (Model DPT-200, lot#1101991, Midvale, UT, USA) via water columns from venogram balloon tipped catheters (Attain 6215, Medtronic, Inc.).
High-resolution Olympus commercial endoscopes (Model 1V8200T, Model 1V8420, Center Valley, PA, USA) were then placed within these heart and/or lungs to capture functional anatomy. To our knowledge, these are the first images of the pulmonary veins and arteries within the lung of functioning human heart-lung blocs.
Results & Discussion
Using this experimental approach, eighteen swine and five human heart-lung blocs were successfully reanimated. Hemodynamic functioning of these in vitro reanimated specimens was augmented by the delivery of inotropic agents (1.5 mg doubutamine) and/or by increased dosing with extracellular calcium (3.5 mg calcium chloride). Prior to heart recovery, the mean heart rate and blood pressure for the swine were: 91±13 beats per minute and 105/56±13/9 mmHg, respectively. Table 23 provides partial cardiac medical histories for the organ donors from which the human hearts were recovered. Table 24 provides the relative hemodynamic performance data for these reanimated heart-lung preparations. These data points are calculated as 5 minute averages at the 1 hour time point post defibrillation.
Table 23. Summary of donor information and hemodynamic status prior to organ recovery Human Specimens Specimen Gender Age (yrs) Weight (kg) Cause of Death HR (bpm) BP (mmHg) CVP (mmHg)
HH 277 M 60 113.4 Head trauma 71 105/61 15 HH 284 F 78 54.4 CVA 103 118/70 11 HH 291 F 58 114.7 CVA 92 100/50 12 HH 295 M 34 68.0 Cardiac arrest 92 130/75 - HH 308 F 36 53.0 CVA, previously transplanted 87 97/71 10 Average 53.2 80.7 89.0 110/65 12 Standard Dev. 18.4 31 11.6 14/10 2.2
BP=blood pressure; HR=heart rate; CVA= cerebrovascular accident; CVP=central venous pressure
Table 24. Hemodynamic performance of each reanimated heart/lung bloc specimen
Swine Specimens Specimen HR (bpm) LVSP (mm Hg) LVEDP (mm Hg) +dLVP/dt (mm Hg/s) -dLVP/dt (mm Hg/s) Tau Lung 1 95.8 91.2 12.4 982.8 -903.0 31.2 Right 2 91.0 25.0 -2.0 430.8 -343.8 36.2 Right 3 100.0 77.0 -4.0 961.0 -462.0 30.0 Right 4 81.7 73.5 2.3 772.3 -354.5 37.7 Right 5 91.8 85.7 1.3 927.0 -509.8 33.2 Right 6 99.5 62.8 11.3 574.0 -435.0 30.2 Right 7 55.8 82.0 5.7 600.2 -358.2 63.0 Right 8 90.3 79.3 -4.3 842.5 -618.0 33.5 Left 9 92.7 75.7 12.0 623.3 -771.7 32.5 Left 10 102.5 67.5 1.2 637.8 -513.2 29.7 Right
11 76.7 101.7 10.7 786.5 -501.5 39.7 Right 12 123.3 76.3 1.3 729.0 -624.5 26.0 Right 13 124.8 58.7 2.7 762.5 -532.7 24.0 Right 14 84.5 87.2 0.7 888.8 -646.0 54.0 Right 15 114.0 91.7 -2.7 922.3 -808.0 27.5 Right 16 105.0 70.8 0.0 607.7 -446.3 28.8 Right 17 101.3 56.5 3.8 529.8 -317.7 29.8 Right 18 91.2 75.7 7.0 618.3 -810.2 33.0 Right Average 95.7 74.3 3.3 733.2 -553.1 34.4 Standard Dev. 16.3 17 5.4 163.6 177.6 9.7 Human Specimens HH 277 85.8 65.7 -7.3 624.5 -475.5 37.2 Right HH 284 81.2 79.5 1.7 848.2 -377.7 37.5 Right HH 291 70.3 53.3 8.0 341.3 -273.7 45.3 Both HH 295 81.3 72.5 4.2 415.7 -343.0 37.7 Right HH 308 57.2 73.0 0.0 963.0 -469.8 56.2 Right Average 75.2 68.8 1.3 638.5 -387.9 42.8 Standard Dev. 11.6 9.9 5.7 268.1 86 8.2
HR=heart rate; LVSP=left ventricular systolic pressure; LVEDP=left ventricular end- diastolic pressure; +dLVP/dt= maximal positive derivative of left ventricular pressure with respect to time; -dLVP/dt= maximal negative derivative of left ventricular pressure with respect to time
It should be noted that one of the early reanimated swine heart-lung specimens (#2) elicited poor hemodynamic performance from the beginning of reanimation. We suspect that injury occurred during isolation and/or that emboli caused poor coronary perfusion. Additionally, recorded data from several hearts elicited negative values for end-diastolic
pressures; we suspect that this is due to a vacuum or syphoning effect, potentially occurring in the current system modification to incorporate the lungs.
Interestingly, compared to our long-term experience with lone heart reanimation using endoscopes, a large degree of remaining particulate and blood within the lung complicated our initial imaging during certain studies. Therefore, more frequent buffer changes were required to obtain clear, high-fidelity images and video.
The main benefit of this model is the maintenance of proper pulmonary ostia and vessel anatomies. A selected anatomical image series of a videoscope being retracted from either the pulmonary arteries or veins is shown in Figure 36; a corresponding video can be accessed via supplemental materials (Supplemental Video) or online at http://www.vhlab.umn.edu/atlas. The study of cryoballoon ablation procedures motivated much of the development of this model, and a series of cryoballoon procedures have been performed, as viewed from within the vein as displayed in Figure 37, panel C. The Supplemental Video of the functioning pulmonary arteries and veins gives the reader an appreciation of the truly dynamic nature of these vessels, which are usually thought to be relatively passive structures. To the authors’ knowledge, this report has provided first time dynamic images of the pulmonary vasculature during normal cardiac function in both reanimated human and swine heart-lung blocs. This model provides a unique in vitro approach for the study of medical therapeutics from both human and large mammalian heart-lung specimens.
Figure 36. Image series obtained from reanimated human heart-lung bloc 284 (A,B) and 277 (C,D). Series shows the path through the distal pulmonary arteries and veins, respectively. The corresponding fluoroscopic images (B,D) in each case show the relative locations of the videoscopes (A,C). A video of the journey through the vasculature can be viewed as well (see online Supplementary Video).
Figure 37. Time series of images from human heart 277. Series shows tricuspid valve closure from the right ventricle (A) and right atria (B). Images are displayed 1/15th per second apart in time. Panel C displays ice formation on the distal portion of a cryoballoon ablation catheter (Artic Front, Medtronic, Inc., Minneapolis, MN) as seen from within the pulmonary vein. The images are spaced post-ablation 30 seconds, 1, 2, and 3 minutes apart.
In a similar embodiment (i.e., without the lungs), this reanimated heart model has been utilized in numerous cardiac studies. In the electrophysiologic area, these studies have included the use of endocardial noncontact mapping, pacemakers, defibrillators, leads, and catheters [155]. The Visible Heart® model has also been employed to study the dynamic nature of valves and transcatheter valve deployment [156]. Importantly these methodological approaches also allow for use of echocardiography and fluoroscopy to guide procedures, such as for comparative imaging [157]. Most recently, this approach has proven to be quite valuable for the study of novel cardiac treatments, such as leadless pacing devices [158]. Nevertheless, the addition of a lung(s) to this paradigm still allows for any of the prior studies to be conducted, but may in turn reduce the number of hemostasis valve access points that were previously available.
Our continued use and enhancement of Visible Heart® methodology has also facilitated the creation of an open-access educational website, The Atlas of Human Cardiac Anatomy (http://www.vhlab.umn.edu/atlas) [159]. The anatomical images and videos on this website are free to download and use for presentations and teaching, however we request that proper citations be used. In other words, the images and/or comparative imaging of functional cardiac anatomies are of high value in teaching the nuances of cardiac anatomy, especially of active, complex structures such as valves. It should be noted that this website also provides instructional tutorials on cardiac anatomy and physiology, as well as full cadaveric thoracic cavity dissections. Finally, a cardiac device tutorial is also available, which has been well noted as being beneficial in explaining therapies to patients.
The model described here is not without limitations, as is true with all in vitro systems. Despite supersaturating the buffer with oxygen, there remains a significant difference in
the oxygen content of the buffer compared to blood. For this reason, the function of the heart slowly declines over time from the initial reanimation. Yet reasonable physiologic function to perform the aforementioned investigations is generally elicited for up to 4 to 8 hours, based on our previous experiences. It is possible that the addition of the lungs may extend the functionality, which we tend to believe at this point, but need more data to substantiate. Most recently, we are employing a full anesthesia suite ventilator to more closely control the ventilation parameters (e.g., provide positive end expiratory pressure, PEEP). In such experimentation, one also needs to consider that although it is also known